The long range goals of our 33-year biophysical research program have been to: (a) develop stereochemical theories for ligand capture and bond formation in animal hemoglobins (Hb) and myoglobins (Mb), and (b) determine the mechanism of how these proteins, microbial single domain globins, and flavohemoglobins oxidize nitric oxide to nitrate. Many of the specific aims associated with these goals have been achieved. We and others have identified the roles of specific amino acids, structural motifs, and stereochemical effects in regulating O2 affinity, ligand discrimination, rates of ligand binding, and NO dioxygenation, using mammalian Mb and human HbA as model systems. We propose to conclude our biophysical studies of the functional properties of hemoglobins and myoglobins in the next grant period by: (1) Verifying the distal histidine (E7) pathway for ligand entry and exit by completing time-resolved X-ray crystallography and FTIR studies of key His(E7) and Phe(CD4) mutants of sperm whale Mb and then evaluating if the E7 pathway is affected by alternative quaternary structure;(2) Testing the applicability of our kinetic and equilibrium ligand binding mechanisms in two single domain animal (Ascaris suum and Cerebratulus lacteus) and two microbial (Aquifex aeolicus and Bacillus anthracis) hemoglobins, which contain markedly altered distal pocket structures (TyrB10/Gln or ThrE7) and, in some cases, an apolar tunnel that may represent an alternative route for ligand entry and exit;and (3) Determining the mechanism for NADH/O2 driven NO dioxygenation (NOD) by E. coli flavohemoglobin and a series of fungal (Candida albicans and Aspergillus fumigatus) flavohemoglobins, which catalyze this reaction as part of a defense mechanism against host-induced nitrosative stress. In the latter case, the NO dioxygenase activities of the microbial flavoHbs will be correlated with those observed for mammalian Mbs and Hbs to identify the biochemical and biophysical principles involved in this crucial physiological reaction. In both classes of globins, NO reacts with bound O2 to produce nitrate directly, without the production of toxic reactive nitrogen or oxygen species. In the case of muscle Mb and red cell Hb, this NO scavenging reaction protects striated muscle and other aerobic tissues from inhibition of mitochondrial respiration during inflammation, sepsis, and nitric oxide inhalation. Our proposed studies will provide: (a) alternative biophysical mechanisms for O2 stabilization, rapid ligand binding, and NO dioxygenation;(b) insight into the evolution of globin structures and functions, from Archaea to Bacteria to Eucarya;and (c) a framework for inhibiting the activities of globins from pathogenic organisms (Candida albicans, Aspergillus fumigatus, Bacillus anthracis, and Ascaris suum).